Developing ethyl lauroyl arginate antimicrobial films to combat Listeria monocytogenes in cured ham
Introduction
Plastics pose a serious pollution problem globally, especially on the seas where its impact is higher. The amount of plastic in the oceans has been estimated to be around 75–199 million tons and it is supposed to double by 2030, affecting the coastlines and marine fauna greatly (UNEP, 2021). Hence, the development of biodegradable packaging is a priority to reducing this environmental problem because packaging produce around 60% of the plastic waste in the EU, mainly through single use plastics (European Comission, 2018). One packaging alternative involves the use of carboxymethyl cellulose (CMC), an anionic linear polysaccharide derived from water-soluble cellulose. Cellulose is the most abundant renewable and biodegradable polymer on earth, and has several advantages such as inexpensiveness, low density, non-toxicity and versatility (Huang & Wang, 2022), which makes CMC, an easily obtained cellulose-derivative, a great sustainable and cheap option to develop biodegradable films. CMC is currently one of the most common derivatives used in edible-film preparation because, when cross-linked with citric acid its resistance to water increases and its swelling ability decreases (Dashipour et al., 2015; Kanatt & Makwana, 2020; Tongdeesoontorn et al., 2011). Therefore, films developed using these biopolymers might be used in foods with low water activity to increase their stability and durability.
In recent years, low-water activity or low moisture content foods have been increasingly associated with food product recalls and foodborne outbreaks due to contamination by human and zoonotic foodborne pathogens, such as Salmonella spp., Listeria monocytogenes, Bacillus cereus, Clostridium botulinum, and Verotoxigenic Escherichia coli (i.e., E. coli O157:H7 and other Shiga toxin-producing strains of E. coli) (Gurtler et al., 2014). For instance, Listeria monocytogenes, a pathogenic bacterium with the ability to form biofilms, is prone to acutely contaminate ready-to-eat (RTE) foods, with serotype 1/2c being one of the most dangerous usually found in meat processing factories (Alía, Andrade, Córdoba, et al., 2020). This particular bacterium is responsible for producing one of the most dangerous bacterial infections, listeriosis, which results in high morbidity and mortality rates (17.6% in 2019) compared with other foodborne pathogens, especially in pregnant women, the elderly and the immunosuppressed. Verotoxigenic Escherichia coli is also a hazardous RTE food pathogen in meat products and cheese, with E. coli O157:H7 representing one of the top five serotypes. Prevalence of L. monocytogenes and Verotoxigenic E. coli in RTE products of pig meat origin, like cured ham, was found to be 2.1% and 1.1%, respectively (European Food Safety Authority, 2021). Microbiological cross-contamination in cured ham is the main problem when it comes to L. monocytogenes and Verotoxigenic E. coli as it can occur by direct contact with personnel, aerosols, contaminated utensils and equipment, especially during the stages of deboning, compressing and slicing (Aecosan, 2014; Alía, Andrade, Rodríguez, et al., 2020). For this reason, it is necessary to develop antimicrobial active packaging with the ability to interact directly with the food product, allowing to improve food safety by limiting bacterial growth in RTE foods.
An effective antimicrobial against bacteria is ethyl lauroyl arginate (LAE, E−243), a cationic surfactant compound approved as a food additive and generally recognized as safe (GRAS) by the European Union (EU) and the Food and Drug Administration (FDA) within the United States. It is used in the EU as a food preservative in heat-treated meat products (Becerril et al., 2013; Nerín et al., 2016; European Parliament and the Concil of the European Union, 2008). However, LAE is soluble in water, which results in its release and migration from the package to the food, hindering its long-term antimicrobial use on foods (Silva et al., 2019). In addition, its liberation also provokes that the acceptable daily intake (ADI) of 0.5 mg/kg body weight can be reached or surpassed easily in toddlers and children (Younes et al., 2019). Hence, new strategies to reduce its availability and water solubility are in demand.
We hypothesized that one such strategy to increase the insolubility of LAE in water would be through the complexation of LAE with polyoxometalates (POMs), which are a diverse class of nanoscale molecular metal oxides (Cronin & Müller, 2012). They form negatively charged aggregates of transition metals (mainly vanadium, molybdenum and tungsten) characterized by a wide and versatile range of physicochemical properties (Gumerova & Rompel, 2020). Typically, their anionic nature is useful to create complexes with cationic compounds (Song & Tsunashima, 2012). In addition, their redox characteristics allow their use in a variety of applications, from catalysis to medicine, since they can be supported by a wide variety of surfaces, such as polymers, and they have also been described to have antimicrobial properties (Bijelic et al., 2018). Moreover, POMs can be combined with functional cations to afford multifunctional compounds. Such POM anion-functional cation complexes have recently been demonstrated to be highly effective antimicrobial compounds and antimicrobial surface coatings, like the polyoxometalate-ionic liquids K8SiW11O39 has the ability to form (Enderle et al., 2022; Kubo et al., 2017; Misra et al., 2018). Regarding their toxicity, polyoxotungstates have been shown to be toxic against human primary peripheral blood mononuclear cells at concentrations over 79.5 μM (Rhule et al., 1998). While there are no studies of acceptable daily intake (ADI) of polyoxotungstates, results have shown the oral median lethal dose (LD50) to be higher than 2000 mg/kg (NICNAS, 1996). To this end, we reasoned that the complexation of cationic LAE with anionic POMs could generate an antimicrobial additive and, furthermore, we reasoned that such complexes could be embedded into biodegradable antimicrobial RTE packaging and retain their broad-spectrum antimicrobial properties.
Here we show how a cation exchange reaction of the monolacunary polyoxotungstate K8SiW11O39 with LAE forms a hybrid antimicrobial POM-LAE complex. Afterwards, POM-LAE can be added to cross-linked CMC to produce composite POM-LAE-CMC antimicrobial films (Fig. 1). Additionally, we demonstrate how the POM-LAE-CMC composite film can be used as proof-of-concept active packaging for cured ham.
Section snippets
K8[SiW11O39] (POM)
The synthesis was a modification of the literature procedure (Tézé et al., 1990). Na2WO4⋅4H2O (182 g, 0.55 mol) was dissolved in distilled water (300 mL) and the solution was heated to reflux. Aqueous 4 M HCI (165 mL) was added dropwise under vigorous stirring to the heated solution over a period of 30 min. Crucially, any precipitate (tungstic acid) formed was allowed to re-dissolve before further acid addition. An aqueous solution of Na2SiO3∙5H2O (11 g, 50 mmol in 100 mL distilled water) was
Synthesis and characterization of POM-LAE
POM-LAE, a white powder with low solubility in water, was synthesized using a cation metathesis route and was obtained in near quantitative yield. The sample composition and purity were confirmed by FT-IR-spectroscopy and thermogravimetric analysis (TGA). FTIR-ATR of the POM-LAE showed the specific vibrations of the polyoxotungstate and LAE molecular components (Fig. 2). The characteristics wavelength vibrations of LAE were reported at 3500-3000 cm−1 corresponding to N–H bond in the amino
Conclusions
Developing easily biodegradable and bio-based packaging materials for RTE foods, is crucial to reduce the ubiquitous plastic pollution in the oceans. The need to develop biodegradable films and improve food safety has led us to develop a new hybrid antimicrobial complex (POM-LAE) and incorporate it into biodegradable CMC films, as a viable composite antimicrobial packaging solution for RTE foods, which are highly prone to bacterial contamination. POM-LAE showed antimicrobial activity against L.
Declaration of competing interest
None.
CRediT authorship contribution statement
Nicolás Gracia-Vallés: Investigation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing, Visualization. Fernando Ruiz-Torrubia: Investigation, Methodology. Scott G. Mitchell: Conceptualization, Investigation, Writing – review & editing, Resources. Cristina Nerín: Conceptualization, Writing – review & editing, Supervision, Funding acquisition. Filomena Silva: Conceptualization, Investigation, Methodology, Writing – review & editing, Resources, Funding
Acknowledgments
This work was supported by the Spanish Ministerio de Ciencia e Innovación (RTI2018-097805-B-I00 and PID2019-109333RB-I00), the Gobierno de Aragón (Project LMP49-18) and the Centre for the Development of Industrial Technology (1061/2020). N.G. acknowledges a fellowship from the Universidad de Zaragoza and Université de Pau et des Pays de l’Adour.
The authors would also like to acknowledge the use of the Advanced Microscopy Laboratory and the Servicio General de Apoyo a la Investigación-SAI, both
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